Actin and myosin are involved in a host of contractile events in nonmuscle cells including cytokinesis, phagocytosis and cell locomotion. The regulation of the interaction of these two proteins is critical to normal cell function. There are at least two possible regulatory systems involved in this. One of these is the myosin light chain kinase dependent phosphorylation of myosin. This kinase is regulated by calcium and calmodulin. Myosin must be phosphorylated in order to be activated. The other system involves caldesmon, an actin- and calmodulin-binding protein which can inhibit myosin's activity in vitro. The inhibition can be reversed by calmodulin binding to caldesmon. Intact platelet caldesmon and a carboxyl-terminal fragment prepared by recombinant DNA techniques and expressed in E. coli inhibit the sliding of fluorescently-labeled actin filaments over myosin bound to a surface in an in vitro motility assay. The amino-terminus of caldesmon does not inhibit actin filament sliding. Caldesmon is a substrate for various kinases and has been shown to be phosphorylated during smooth muscle contraction. It is also phosphorylated when human platelets are treated with prostaglandins at sites phosphorylated in vitro by cAMP-dependent protein kinase. We have examined the site phosphorylated on caldesmon by this kinase and find that they are in the amino-terminal two-thirds of the molecule.